Identification of an immunomodulating ... - Wiley Online Library

Cellular Microbiology (2012) 14(6), 902–913

doi:10.1111/j.1462-5822.2012.01765.x First published online 24 February 2012

Identification of an immunomodulating metalloprotease of Pseudomonas aeruginosa (IMPa) Bart W. Bardoel,* Dennis Hartsink, Mignon M. Vughs, Carla J. C. de Haas, Jos A. G. van Strijp and Kok P. M. van Kessel Department of Medical Microbiology, University Medical Center Utrecht, 3584CX Utrecht, The Netherlands. Summary Phagocytosis by neutrophils is the essential step in fighting Pseudomonas infections. The first step in neutrophil recruitment to the site infection is the interaction of P-selectin (on endothelial cells) with P-selectin glycoprotein ligand-1 (PSGL-1) on neutrophils. Pseudomonas aeruginosa secretes various proteases that degrade proteins that are essential for host defence, such as elastase and alkaline protease. Here we identify PA0572 of P. aeruginosa as an inhibitor of PSGL-1 and named this secreted hypothetical protease immunomodulating metalloprotease of P. aeruginosa or IMPa. Proteolytic activity was confirmed by cleavage of recombinant and cell-surface expressed PSGL-1. Functional inhibition was demonstrated by impaired PSGL-1-mediated rolling of IMPa-treated neutrophils under flow conditions. Next to PSGL-1, IMPa targets CD43 and CD44 that are also involved in leucocyte homing. These data indicate that IMPa prevents neutrophil extravasation and thereby protects P. aeruginosa from neutrophil attack.

Introduction Pseudomonas aeruginosa is a Gram-negative bacterium that is commonly found in soil and water. In humans, it acts as an opportunistic pathogen causing severe chronic infections in cystic fibrosis patients (Costerton et al., 1999). P. aeruginosa colonizes the cystic fibrosis lung where it forms biofilms that are insensitive to antimicrobial treatment (Whiteley et al., 2001). In healthy hosts, the innate immune system very rapidly and efficiently kills invading microorganisms. Gram-negative bacteria are lysed by complement via the membrane attack complex; Received 9 September, 2011; revised 21 December, 2011; accepted 23 January, 2012. *For correspondence. E-mail b.w.bardoel@ umcutrecht.nl; Tel. (+31) 88 7556525; Fax (+31) 88 7555863.

however, many P. aeruginosa strains are insensitive to complement-mediated killing (Young and Armstrong, 1972). Furthermore, this bacterium secretes several proteins that contribute to virulence and assist to circumvent different branches of the innate immune system (Matsumoto, 2004). The proteases elastase and alkaline protease of P. aeruginosa degrade components of the complement system (Hong and Ghebrehiwet, 1992), and immunoglobulins (Wolz et al., 1991). Expression of both proteases as well as other pseudomonal proteins are regulated via the transcriptional regulatory protein LasR that belongs to the quorum sensing system las (Gambello et al., 1993; Nouwens et al., 2003). This system contributes to P. aeruginosa virulence in vivo (Tang et al., 1996). Production of pro-inflammatory cytokines and chemokines after recognition of conserved bacterial structures by the innate immune system attracts leucocytes to the site of infection. These inflammatory mediators induce the expression of E- and P-selectin (Vestweber and Blanks, 1999). P-selectin glycoprotein ligand-1 (PSGL-1) is the major ligand for P-selectin (Moore et al., 1992; Somers et al., 2000); however, PSGL-1 also interacts with E-selectin (Xia et al., 2002). Transient interactions between PSGL-1 and these two selectins initiate rolling of leucocytes over the endothelium, the first step in recruitment of leucocytes to the site of infection. PSGL-1 is expressed on most leucocytes including lymphocytes, monocytes and neutrophils. It is a heavily glycosylated transmembrane protein of 120 kD and expressed as a disulfide-linked homodimer on cells. PSGL-1 contains three N-linked glycans, sialylated and fucosylated O-linked glycans, which often terminate with a sialyl Lewis X oligosaccharide (Epperson et al., 2000). Proper glycosylation of PSGL-1 is required for interaction with P-selectin (Carlow et al., 2009). The glycoprotein CD44 expressed on leucocytes binds E-selectin and can cooperate with PSGL-1 to initiate rolling of neutrophils over the endothelium (Katayama et al., 2005). The extracellular matrix component hyaluronan is another ligand of this type I transmembrane cellsurface receptor (Jiang et al., 2007). Cell adhesion and migration by CD44 play a role in different biological processes like lymphocyte activation and tumour-cell migration. In addition to CD44, the heavily glycosylated leukosialin receptor (CD43) expressed on various

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cellular microbiology

PSGL-1 inhibition by P. aeruginosa leucocytes is also involved in cell adhesion (Fukuda and Tsuboi, 1999). The O-glycan structures on CD43 differ between cell types, lymphocytes contain a low and neutrophils a high molecular weight O-glycan structure. Some studies (Matsumoto et al., 2005), but not others (Carlow and Ziltener, 2006), suggest that CD43 also binds to E-selectin on activated T cells. CD43, CD44 and PSGL-1 all contain mucin-type O-glycans, which are essential for the interaction with E-selectin (Yago et al., 2010). Bacteria exploit several strategies to interfere with various components of the innate immune system (Rooijakkers et al., 2005). The secreted protein staphylococcal superantigen-like 5 (SSL5) of Staphylococcus aureus binds to PSGL-1 in a sialyl Lewis X-dependent way (Bestebroer et al., 2007) and to other proteins that contain this carbohydrate structure (Bestebroer et al., 2009). SSL5 inhibits rolling of neutrophils on activated human umbilical vein endothelial cells (HUVECs). In this study, we screened the secretome of P. aeruginosa for the presence of proteins that interfere with the P-selectin/PSGL-1 interaction. Prevention of anti-PSGL-1 monoclonal antibody binding to leucocytes treated with concentrated P. aeruginosa supernatant confirmed the existence of a PSGL-1 inhibitor. Fractionation of the supernatant followed by mass spectrometry analysis revealed that the hypothetical metalloprotease PA0572 exerts proteolytic activity for PSGL-1 and three other leucocyte cell-surface receptors. Results Isolation of a PSGL-1 inhibitor from P. aeruginosa supernatant Pseudomonas aeruginosa supernatant was monitored for the presence of potential inhibitors of the leucocyte cellsurface receptor PSGL-1 (CD162). Treatment of lymphocytes with concentrated supernatant of P. aeruginosa decreased the binding of a CD162 antibody. Activity was maintained after additional purification by ion-exchange and size-exclusion chromatography (Fig. 1A). Fractions that inhibited CD162 antibody binding corresponded with a 100 kD band on SDS-PAGE (Fig. 1B). The protein of interest was identified by mass-spec finger printing as PA0572, a hypothetical protein of P. aeruginosa. Two other bands of 60 and 40 kD that almost matched with the PSGL-1 inhibitory activity were identified as an intracellular protein involved in energy metabolism and the flagellar hook-associated protein FlgK respectively. Immunomodulating metalloprotease of Pseudomonas aeruginosa (IMPa) PA0572 was previously identified in the secretome of P. aeruginosa (Nouwens et al., 2003), under regulation of © 2012 Blackwell Publishing Ltd, Cellular Microbiology, 14, 902–913

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Fig. 1. Isolation of a PSGL-1 inhibitor from P. aeruginosa supernatant. Supernatant of overnight cultured P. aeruginosa (PAO1) was precipitated with ammonium sulfate and separated by ion-exchange and size-exclusion chromatography. A. Leucocytes were treated with concentrated sample before (conc.) and after separation and fractionation (1–12) by size-exclusion chromatography for 30 min at 37°C. After washing, cells were stained with PE-labelled anti-PSGL-1 (KPL1) for 30 min at 4°C. The geometric mean fluorescence of lymphocytes was measured, by gating for forward- and side-scatter properties. Data are depicted as relative values compared with PSGL-1 staining of untreated cells. B. The same fractionated samples were loaded on a SDS-PAGE gel and stained with Coomassie. The 100 kD band corresponded with inhibitory activity and was together with the 60 kD and 40 kD analysed by mass spectrometry.

LasR. This regulator is involved in expression of several virulence factors of P. aeruginosa. Sequence analysis revealed that PA0572 contains a zinc protease motif (GESHELGHNL), which is a conserved proteolytic domain found in zinc metalloproteases. A BLAST search revealed that this hypothetical protease is conserved (over 90%) in all eight sequenced P. aeruginosa strains and absent in other Pseudomonas species. A large-scale purification with an additional ionexchange step was performed to obtain sufficient pure protein for functional experiments (Fig. 2A). PSGL-1-Fc was incubated with PA0572 to verify its proteolytic activity, resulting in several degradation products (Fig. 2B). Proteolytic activity was blocked in the presence of EDTA, which inhibits metalloproteases. Purified PA0572 dosedependently decreased binding of anti-CD162 to leucocytes (Fig. 2C–E). Inhibition was more pronounced on lymphocytes (Fig. 2C) in comparison with neutrophils and

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Fig. 2. IMPa dose-dependently cleaves PSGL-1. A. Purity of PA0572 after large-scale purification from P. aeruginosa supernatant with an additional monoQ separation step. B. Proteolytic activity of PA0572 was confirmed by incubation of PSGL-1-Fc with 3 mg ml-1 PA0572 ⫾ 10 mM EDTA. Samples were analysed by SDS-PAGE and Coomassie staining. C–E. Leucocytes were treated with various concentration of PA0572 ⫾ 10 mM EDTA for 30 min at 37°C or at 4°C. After washing, cells were stained with PE-labelled anti-PSGL-1. The relative geometric mean of (C) lymphocytes (D) monocytes and (E) neutrophils was analysed by gating for forward- and side-scatter properties. F. Time-dependent inhibition of PA0572. Leucocytes were incubated with 3 mg ml-1 PA0572 at 37°C and after 1, 3, 10, 20 or 30 min 10 mM EDTA was added to block proteolytic activity of PA0572, followed by anti-PSGL-1 staining. Data represent the relative geometric mean (compared with control) ⫾ SEM of three independent experiments. B–E. Statistics were calculated between IMPa-treated cells and IMPa-treated cells in the presence of EDTA.

monocytes (Fig. 2D and E). In addition, activity was blocked when incubated at 4°C or in the presence of EDTA. This demonstrates that PA0572, as predicted by sequence analysis, acts as a zinc metalloprotease and shows that proteolytic degradation is responsible for decreased antibody detection of PSGL-1. Inhibition was time-dependent and observed within 3 min after addition of PA0572 (Fig. 2F). Because PSGL-1 plays an essential role in leucocyte homing and extravasation, we named PA0572, Immunomodulating Metalloprotease of Pseudomonas aeruginosa (IMPa).

smaller extent on monocytes and neutrophils (only inhibition of CD43). Decay accelerating factor (CD55) recognition was inhibited on all three cell types with about twofold. The other tested surface receptors were not affected by IMPa (Figs 4A and S1). The three additional inhibited (CD43, CD44, CD55) and two unaffected (CD46 and CD31) in the cell-surface receptor screening were further investigated at various protease concentrations. The dose–response curves of PSGL-1 (CD162) and CD43 on lymphocytes were similar (Fig. 4B), while CD43 inhibition on monocytes (Fig. 4C)

Bacterial supernatant lacking IMPa To verify that secreted IMPa from P. aeruginosa is responsible for PSGL-1 inhibition, we performed experiments with IMPa transposon mutants obtained from the P. aeruginosa transposon library (Jacobs et al., 2003). Integration of the transposon in the impa gene was confirmed by PCR (data not shown). Concentrated bacterial supernatant, partially purified by size-exclusion chromatography of two IMPa mutants strains failed to inhibit PSGL-1 recognition, whereas wild type showed activity (Fig. 3A). Furthermore, concentrated supernatant of IMPa mutant strain showed a comparable pattern on SDSPAGE as wild-type except the absence of a 100 kD band (Fig. 3B), which is the size of IMPa in the corresponding active fractions. These data demonstrate the IMPa is responsible for the cleavage of PSGL-1 and corresponds with the presence of the impa gene. Protease specificity Elastase and alkaline protease of P. aeruginosa have both a broad specificity and cleave various host proteins that are involved in antimicrobial defence (Matsumoto, 2004). IMPa was identified by screening for PSGL-1 inhibitors; however, its activity on other cell-surface receptors was not determined. To address its specificity, we treated leucocytes with 3 mg ml-1 IMPa and stained with a panel of antibodies that recognize different cellsurface receptors involved in innate immunity. Antibody recognition was clearly impaired for CD43 and CD44 (Fig. 4A) on lymphocytes (four- to eight-fold) and to a © 2012 Blackwell Publishing Ltd, Cellular Microbiology, 14, 902–913

Fig. 3. Partially purified supernatant of a P. aeruginosa IMPa mutant lacks activity for PSGL-1. Supernatant of P. aeruginosa wild-type PAO1 and the IMPa transposon mutant was concentrated and loaded on a Superdex 75 column and fractions of 0.5 ml were collected. A. Leucocytes were pretreated with gelfiltration fractions of the wild-type and IMPa mutant for 30 min at 37°C and were stained with PE-labelled anti-PSGL-1. Untreated cells were stained with isotype control and anti-PSGL-1. Lymphocytes were gated according to forward- and side-scatter properties and data are expressed as relative geometric mean values compared with PSGL-1 staining of untreated cells. B. Gelfiltration fractions of PAO1 with inhibitory activity and the corresponding fractions of the IMPa mutant were separated by SDS-PAGE and stained with Coomassie. Arrow indicates the size of IMPa.

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Fig. 4. IMPa specificity. A. Leucocytes were pretreated with 3 mg ml-1 IMPa for 30 min at 37°C and stained with a panel of fluorescently labelled monoclonal antibodies that recognize different cell-surface receptors of leucocytes. Fold inhibition was calculated by dividing the fluorescence (geometric mean) of untreated cells by that of treated cells. Data represent the mean value ⫾ SEM of three independent experiments. B–D. Leucocytes were incubated with different concentrations of IMPa for 30 min at 37°C, followed by incubation with the antibodies: CD162-PE, CD44-PE, CD43-FITC, CD55-APC, CD46-PE and CD31-FITC. Lymphocytes (B), monocytes (C) and neutrophils (D) were gated according to forward- and side-scatter properties. Relative fluorescent values were calculated by comparison to untreated cells and represent mean ⫾ SEM of three independent experiments. For lymphocytes (B) inhibition of CD43 is statistical significant for 0.3–10 mg ml-1, CD44 1–10 mg ml-1, CD55 10 mg ml-1 and CD162 0.1–10 mg ml-1 IMPa. For monocytes (C) inhibition of CD44 is statistical significant at 10 mg ml-1, CD44 at 3–10 mg ml-1, and CD162 at 1–10 mg ml-1 IMPa. In the rest of the figure statistical significance is indicated. © 2012 Blackwell Publishing Ltd, Cellular Microbiology, 14, 902–913

PSGL-1 inhibition by P. aeruginosa and neutrophils (Fig. 4D) was less pronounced compared wtih PSGL-1. CD44 and CD55 inhibition was observed on IMPa-treated lymphocytes, whereas on neutrophils and monocytes only at high concentrations some inhibition could be detected. At the highest dose, IMPa showed no inhibition of CD46 and CD31 staining. Although IMPa inhibition was not restricted to PSGL-1, the protease showed activity for only a small number of tested cellsurface receptors. IMPa cleavage of CD43 and PSGL-1 To further analyse the cleavage site of IMPa, we incubated recombinant human PSGL-1-Fc with IMPa and performed Western blotting, with three antibodies that recognize different parts of PSGL-1. KPL1 (used for screenings assay) and PL1 recognize the N-terminal domain of PSGL-1 and PL2 recognizes a more proximal domain of PSGL-1 (Epperson et al., 2000) (Fig. 5A). The N-terminal epitope was removed after IMPa treatment of recombinant PSGL-1-Fc (Fig. 5B), in contrast PL2 still recognized two degradation products of 50 and 100 kD (Fig. 5B). Flow cytometric analysis of PSGL-1 cleavage on lymphocytes revealed that the PL2 epitope remained available on IMPa-treated cells (Fig. 5C). These results demonstrate that IMPa removes the N-terminal domain of cell bound PSGL-1, which is essential for the interaction with P-selectin. Next to PSGL-1, IMPa efficiently inhibited CD43 antibody binding (IC50 of 0.2 mg ml-1) especially to lymphocytes (Fig. 4B). Proteolytic degradation of cell-bound CD43 and PSGL-1 was studied in the supernatant and cell lysates of IMPa-treated lymphocytes. Increasing concentrations of IMPa diminished CD43 and PSGL-1 detection in cell lysates; however, degradation products of both receptors were not detected in the supernatant (Fig. 5D). IMPa activity is not dependent on sialic acids Binding of PSGL-1 to P-selectin is dependent on terminal glycan components that include sialic acid and fucose, typified by the sialyl Lewis X (sLex) determinant. Naïve lymphocytes cannot bind to P-selectin, as they lack fucosyltransferase 7 (FuT7), necessary for proper sLex synthesis (Carlow et al., 2009). Both removal of sialic acids by sialidase and disruption of FuT7 eliminates P-selectin binding. In view of the fact that IMPa cleaves cell-surface receptors more efficient on lymphocytes in comparison with monocytes and neutrophils, we investigated the activity of IMPa after removal of sialic acid by sialidase. Treatment of cells with sialidase was effective as illustrated by impaired binding of anti-CD43, which recognizes a sialidase-sensitive epitope (Fig. 6A). Pretreatment of © 2012 Blackwell Publishing Ltd, Cellular Microbiology, 14, 902–913

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neutrophils and monocytes with sialidase enhanced the IMPa activity towards PSGL1, as binding of anti-PSGL1 was completely lost (Fig. 6A). Removal of sialic acids also clearly enhanced the activity for CD55 on all cell types, whereas cleavage of CD44 was unaffected. Recombinant CD44-Fc, CD55 and PSGL-1-Fc were incubated with IMPa in the presence of sialidase to investigate the effect of sialic acids on proteolytic activity. IMPa degraded PSGL-1 and CD55 efficiently, whereas a relative small part of CD44 was cleaved in the absence of sialidase (Fig. 6B). Removal of sialic acids enhanced CD55 and PSGL-1 cleavage and the molecular weight of CD44 and CD55 degradation products slightly decreased. In conclusion, removal of sialic acids by sialidase seems to enhance the IMPa activity. Functional inhibition by IMPa CD44 expressed on leucocytes interacts with the extracellular matrix component hyaluronan. Binding of recombinant CD44-Fc to a hyaluronan coated surface was inhibited by IMPa (Fig. 7A). To investigate whether IMPa also interferes with PSGL-1/P-selectin interaction, we measured the adhesion of IMPa-treated neutrophils to a P-selectin-coated surface under static conditions. Adhesion of neutrophils was blocked by the anti-PSGL1 monoclonal antibody KPL1. IMPa reduced the adhesion of neutrophils with 50% at 0.1 mg ml-1 and completely abolished adhesion at 3 mg ml-1 protease (Fig. 7B). The impaired interaction of PSGL-1 and CD44 with their ligands suggests that degradation of these receptors by IMPa has functional consequences. In the bloodstream, PSGL-1 initiates rolling of leucocytes by transient interactions with P-selectin on endothelial cells. These conditions were mimicked using a flow chamber in which neutrophils were perfused over glass coverslips coated with P-selectin-Fc or histamineactivated HUVECs. After 5 min, the number of rolling neutrophils was determined by measuring the number of adhered cells per square millimetre. Pre-incubation of neutrophils with anti-PSGL-1 (KPL1) impaired rolling adhesion with at least 80% over a P-selectin-Fc coated surface and HUVECs, demonstrating that rolling was dependent on PSGL-1 (Fig. 7C and D). In the presence of 10 mg ml-1 IMPa rolling adhesion was diminished with 73% on the P-selectin-Fc coated surface and 63% over activated HUVECs (Fig. 7C and D). To check whether IMPa is also produced during pseudomonal infections, we measured the presence of antibodies against IMPa in 22 healthy donors. In all donors we detected antiIMPa IgG (Fig. 7E), indicating that P. aeruginosa secretes IMPa during infection. In conclusion, IMPa potently inhibits binding of PSGL-1 under static and flow conditions.

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Fig. 5. CD43 and PSGL-1 cleavage by IMPa. A. Schematic representation of dimeric PSGL-1 as expressed on cells. The two antibodies PL1 and KPL1 recognize an epitope in the N-terminal domain of the molecule, whereas PL2 detects an epitope in the central part of PSGL-1. B. Western blot analysis of PSGL1 cleavage. PSGL-1-Fc was treated with 0, 0.1, 1 and 10 mg ml-1 IMPa for 30 min at 37°C. Proteins were separated by SDS-PAGE and analysed by Western blotting. After blocking, blots were stained with PL2, PL1 or KPL1, followed by a HRP-labelled goat anti-mouse IgG. C. Leucocytes were pretreated with increasing concentrations of IMPa for 30 min at 37°C and stained with the PSGL-1 antibodies PL1, PL2 and KPL1, followed by incubation with a PE-labelled goat anti-mouse IgG. The relative fluorescence was determined by comparison with untreated cells. Data represent the mean ⫾ SEM of three independent experiments. D. Lymphocytes (CD14 negative leucocytes) were washed with PBS and incubated with 0-0.1-0.3-1-3-10 mg ml-1 IMPa for 30 min at 37°C. Cell supernatant was collected by centrifugation and cell pellet was lysed by the addition of NP-40 lysis buffer for 1 h at 4°C to obtain cell lysate. Samples were separated by SDS-PAGE and transferred to PVDF membrane. Detection was performed with ECL after staining with anti-CD162 (KPL1) and anti-CD43 followed by a HRP-labelled goat anti-mouse IgG.

Discussion Pseudomonas aeruginosa secretes several proteases that degrade proteins of the host immune system and extracellular matrix components. In the present study, we isolated a yet uncharacterized metalloprotease, IMPa (PA0572), which shows proteolytic activity towards four

glycosylated leucocyte surface receptors. Homology search revealed that IMPa is only present in P. aeruginosa and absent in all other Pseudomonas species. IMPa homologues are found in Vibrio and Shewanella that also belong to the gammaproteobacteria. However, the conservation at protein level is rather low, around or below 30%. All these homologues are hypothetical proteins © 2012 Blackwell Publishing Ltd, Cellular Microbiology, 14, 902–913

PSGL-1 inhibition by P. aeruginosa

Fig. 6. Effect of sialidase on IMPa specificity. A. Leucocytes were incubated with 0.2 U ml-1 sialidase from Clostridium perfringens for 45 min at 37°C. Followed by treatment with 1 mg ml-1 IMPa for 30 min at 37°C. Cells were stained with CD43-FITC, CD44-PE, CD162-PE and CD55-APC for 30 min at 4°C. Histograms show binding of the different antibodies to lymphocytes, monocytes and neutrophils that were gated according to forward- and side-scatter characteristics. Histograms represent, untreated cells (black filled histograms), IMPa-treated cells (grey filled histograms) and unstained cells (dotted line). B. Recombinant PSGL-1-Fc, CD44-Fc and CD55 all 50 mg ml-1 were incubated with 3 mg ml-1 IMPa. Next, for sialidase treatment, 50 mg ml-1 NanA was added for 30 min at 37°C. Cleavage was analysed by SDS-PAGE and silver staining.

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containing a signal sequence and a zinc metalloprotease motif. The IMPa-sensitive cell-surface receptors CD43, CD44, CD55 (Lublin and Atkinson, 1989) and PSGL-1 are abundantly O-linked glycosylated, suggesting that IMPa activity is dependent on O-linked glycosylation. The activity of IMPa for CD44 and CD55 is clearly less pronounced than for CD43 and PSGL-1. The same degree of specificity for highly O-glycosylated proteins was described for the secreted metalloprotease of Pasteurella haemolytica. This protease shows high specificity for O-glycosylated cell-surface receptors, including CD43 and CD44, whereas it cannot cleave N-glycosylated proteins or nonglycosylated proteins (Abdullah et al., 1992; Sutherland et al., 1992). The glycosylation status of PSGL-1 differs between leucocytes. In contrast to naive lymphocytes, neutrophils and monocytes express the fucosyltransferase 7, necessary for proper sLex synthesis and P-selectin binding. Activation of lymphocytes induces FuT7 expression, resulting in proper glycosylation of PSGL-1 to interact with P-selectin (Carlow et al., 2009). We show higher activity of IMPa for CD43 and PSGL-1 expressed on lymphocytes in comparison with neutrophils and monocytes. This suggests that additional glycosylation, embodied by the tetrasaccharide sLex, impairs IMPa activity. Indeed, the cleavage efficacy of IMPa for CD55 and PSGL-1 on neutrophils and monocytes, was enhanced after removal of sialic acid by neuraminidase. Thus, a functional sLex moiety seems to hamper IMPa activity. Comparable higher protease activities towards CD43 after removal of sialic acids by neuraminidase are described for pancreatic elastase and V8 protease of S. aureus (Remold-O’Donnell and Rosen, 1990). In contrast to IMPa and the above described proteases, the activity of the secreted metalloprotease of P. haemolytica, which also degrades CD43 and CD44 (Sutherland et al., 1992), was lost upon sialidase treatment (Abdullah et al., 1992). The same has been described for the activity of SSL5 of S. aureus, although SSL5 is not a protease. SSL5 interacts and functionally inhibits PSGL-1 in a sLexdependent way, as desialylation of PSGL-1 completely blocked SSL5 binding and activity (Bestebroer et al., 2007). Therefore, in contrast to IMPa, SSL5 shows activity towards neutrophils and monocytes, but not towards PSGL-1 expressed on naïve lymphocytes. Pseudomonas aeruginosa itself produces a sialidase, which is involved in respiratory infection and biofilm formation (Soong et al., 2006). Mutant strains that lack sialidase fail to colonize the respiratory tract, however, are fully virulent when introduced intravenously. Desialylation of mucosal surfaces increases the adherence and contributes to the colonization of P. aeruginosa (Cacalano et al., 1992). The gene encoding sialidase is among the most conserved and highest expressed in P. aeruginosa

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Fig. 7. Functional effects of IMPa. A. An ELISA plate was coated with 100 mg ml-1 hyaluronan for 1 h at RT. Plate was blocked with 4% skimmed milk in PBS-Tween for 1 h at RT. After washing, CD44-Fc was incubated with IMPa in 1% skimmed milk PBS-Tween and directly added to the 96-wells plate for 1 h at 37°C. After washing, HRP-labelled goat anti-human (1/10.000) in 1% skimmed milk PBS-Tween was added to the wells for 1 h at RT. TMB was used as substrate and the absorbance at 450 nm was determined. Data represent mean ⫾ SEM of three independent experiments. B. An ELISA plate was coated with 3 mg ml-1 P-selectin and blocked with 4% skimmed milk in PBS. Neutrophils were labelled with 4 mM calcein-AM for 20 min at RT. Cells (3 ¥ 105/well) were pretreated with IMPa for 15 min at 37°C before addition to the plate for 15 min at RT. Adherence to P-selectin was determined by measuring the fluorescence after several washes with PBS. Data represent the mean values ⫾ SEM of three independent experiments. C. Neutrophils were treated with 10 mg ml-1 IMPa or KPL1 (anti-PSGL-1) as a positive control for 30 min at 37°C and subsequently perfused over P-selectin-Fc coated glass coverslips with a shear stress of 1.6 dyn cm-2 for 5 min at 37°C. After washing for 1 min, accumulated neutrophils were quantified. D. Isolated HUVECs were cultured on glass coverslips and stimulated with 100 mM histamine for 3 min. Neutrophils were pre-incubated with 10 mg ml-1 IMPa or KPL1 for 30 min at 37°C and subsequently perfused at 0.8 dyn cm-2 for 5 min at 37°C over activated HUVECs. After washing for 1 min, accumulated neutrophils were quantified. Data represent relative accumulation of treated neutrophils compared with control cells and are mean values ⫾ SEM of at least three independent experiments. E. ELISA plates were coated with 1 mg ml-1 IMPa and blocked with 4% BSA. After washing, serial dilutions of serum from 22 healthy donors were added to the IMPA-coated ELISA plate. Anti-IMPa was detected using a HRP-labelled goat anti-human IgG.

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PSGL-1 inhibition by P. aeruginosa strains isolated from cystic fibrosis patients (Lanotte et al., 2004) and increases biofilm formation. Production of sialidase by P. aeruginosa in the respiratory tract can facilitate removal of sialic acids from leucocyte receptors. This will increase the susceptibility of these receptors to subsequent cleavage by IMPa, especially on neutrophils. The secreted proteases elastase, alkaline protease, LasA and protease IV of P. aeruginosa all target multiple proteins (Engel et al., 1998; Matsumoto, 2004; Spencer et al., 2010). The cleavage efficiency widely varies between different substrates of Pseudomonal proteases. At high concentrations, these proteases cleave many different proteins; however, the biological relevance is questionable. IMPa production is regulated via the regulator LasR of the las quorum sensing system (Nouwens et al., 2003). The expression of LasR is dependent on culture conditions (Duan and Surette, 2007). For instance, the expression of LasR and alkaline protease was enhanced in P. aeruginosa grown in the presence of sputum from cystic fibrosis patients. The production of IMPa by P. aeruginosa in overnight culture supernatant was sufficient to successfully purify the protease and in this concentration range IMPa displays proteolytic activity and immune evasive features. Three leucocyte receptors (CD43, CD44 and PSGL-1) that were identified in the IMPa target screenings assay interact with selectins. Removing these receptors form the surface of leucocytes impairs the collective rolling adhesion and consequently transmigration to the site of infection as demonstrated in our rolling experiments with neutrophils. In this way, P. aeruginosa can prevent recruitment and activation of cells of the immune system and thereby increases its survival in the host. These findings suggest a role for IMPa in P. aeruginosa pathogenesis and persistence that can be addressed by studying IMPa knockouts in vivo. Experimental procedures IMPa isolation Pseudomonas aeruginosa (PAO1) was cultured overnight at 37°C in Luria–broth (LB). Supernatant was collected by centrifugation and proteins were precipitated with ammonium sulfate (60% saturation) overnight at 4°C. Precipitated proteins were pelleted, dissolved in distilled water and dialysed against 20 mM sodium phosphate pH 7. Sample was loaded on a DEAE column (GE Healthcare), washed with phosphate buffer and eluted with the same buffer + 1 M NaCl. Active fractions in the antibody competition assay were concentrated using a 30 kD cut-off ultrafiltration device (Millipore) and applied on a Superdex 200 (GE Healthcare) size-exclusion column equilibrated with PBS. Active fractions were diluted five times in phosphate buffer pH 7 and loaded on a MonoQ 5/50 GL column (GE Healthcare). After washing, proteins were eluted with a gradient of 0–0.5 M NaCl. Fractions were analysed for purity using SDS-PAGE and Coomassie (Instant Blue, Expedeon) staining, and proteins of interest were identified by mass spectrometry. © 2012 Blackwell Publishing Ltd, Cellular Microbiology, 14, 902–913

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Antibody competition assay Leucocytes from healthy volunteers were isolated as described (Bestebroer et al., 2007). Cells were incubated with eluted fractions or purified protein diluted in RPMI (Invitrogen) supplemented with 0.05% human serum albumin (HSA; Sanquin) for 30 min at 37°C. Cells were washed with RPMI-HSA and incubated with different antibodies (Supporting information) as described (Bestebroer et al., 2007) for 30 min at 4°C. After washing, cells were analysed using a FACSCalibur (BD biosciences). Neutrophils, monocytes and lymphocytes were selected by gating for forward and side scatter profiles. Where indicated, cells were incubated prior to protease treatment with 0.2 U ml-1 sialidase (Clostridium perfringens; Roche) for 45 min at 37°C.

P. aeruginosa transposon mutants Transposon mutants were obtained from the P. aeruginosa transposon mutant library (Jacobs et al., 2003) and transposon insertion was verified by PCR. Overnight culture supernatant was concentrated using a 30 kD cut-off ultrafiltration device. Concentrated samples were applied on a Superdex 75 (GE Healthcare) column equilibrated with PBS and eluted fractions were analysed in the PSGL-1 (CD162) antibody competition assay.

Cleavage of cell-surface receptors Recombinant PSGL-1-Fc, CD44-Fc or CD55 (50 mg ml-1; R&D systems) were incubated with IMPa in PBS for 30 min at 37°C. Sialidase treatment was performed using 50 mg ml-1 NanA (from Streptococcus pneumonia, kindly provided by G. Taylor) for 30 min at 37°C directly after IMPa treatment. Samples were separated by SDS-PAGE, and proteins were stained with silver. For Western blot analysis, recombinant PSGL-1-Fc (10 mg ml-1) or lymphocytes [CD14 negative leucocytes isolated using the immunomagnetic EasySep CD14 selection kit (Stemcell)], were incubated with IMPa for 30 min at 37°C. Cells were centrifuged and supernatant was collected for analysis. Pelleted cells were lysed in Nonidet P-40 buffer + 10 mM leupeptin and 1 mM 4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride for 1 h at 4°C. Cell debris was removed by centrifugation and supernatant was collected (cell lysate). Samples were separated by SDS-PAGE, transferred to a PVDF membrane, and blocked with 4% skimmed milk in PBS + 0.05% Tween. Next, blots were incubated with anti-CD43 (1/100) or PSGL-1 antibodies PL1, PL2 and KPL1 (0.5 mg ml-1) for 1 h at 37°C. As secondary antibody a HRP-labelled goat anti-mouse IgG (1/10.000, Biorad) was used. Bands were visualized by enhanced chemiluminescence (Amersham).

Hyaluronan/CD44 ELISA A 96-wells plate (Nunc MaxiSorp) was coated with 100 mg ml-1 hyaluronan from rooster comb (Sigma) in 0.1 M sodiumcarbonate buffer pH 9.6 for 1 h at RT. After washing with PBS-Tween, the plate was blocked with 4% skimmed milk in PBS-Tween for 1 h at RT and washed again. CD44-Fc and IMPa were mixed and added to the hyaluronan coated plate for 1 h at 37°C. The plate was washed with PBS-Tween and incubated with HRP-labelled

912 B. W. Bardoel et al. goat anti-human IgG (1/10.000, SouthernBiotech) for 1 h at RT. After washing, the ELISA was developed with TMB as substrate, stopped with H2SO4 and optical density at 450 nm was measured.

Static adhesion assay Static adhesion of neutrophils was performed as described (Bestebroer et al., 2007) with minor modifications. Calceinlabelled cells (3 ¥ 105 ml-1) were incubated with IMPa for 15 min at 37°C in Hanks balanced salt solution with 0.05% HSA, and subsequently added to the recombinant P-selectin (R&D systems) coated plate for 15 min at RT. Adhered cells were quantified after washing the plates several times with PBS using a platereader fluorometer (FlexStation; Molecular Devices) at 450 nm excitation and 530 nm emission.

Neutrophil rolling Rolling experiments with neutrophils were performed as described (Bestebroer et al., 2007). Neutrophils were pretreated with 10 mg ml-1 IMPa for 30 min at 37°C, and subsequently perfused over glass coverslips coated with 10 mg ml-1 P-selectin/Fc (R&D systems) at a shear stress of 1.6 dyn cm-2 at 37°C. HUVECs were stimulated with 100 mM histamine (Sigma) for 3 min to induce expression of P-selectin, and neutrophils were immediately perfused at 0.8 dyn cm-2. Number of adherent cells was visualized using a camera, and recorded images were analysed using the program Optimas 6.1 (Media Cybergenetics Systems).

Detection of IMPa antibodies ELISA plates were coated with 1 mg ml-1 IMPa in PBS for 1 h at 37°C. After washing with PBS-Tween, wells were blocked with 4% BSA for 1 h at 37°C. Plates were washed with PBS-Tween, subsequently serial dilutions of sera were added for 1 h at 37°C. The plate was washed with PBS-Tween and incubated with HRPlabelled goat anti-human IgG (1/10.000, SouthernBiotech) for 1 h at 37°C. After washing, TMB was added as substrate, reaction was stopped with H2SO4, and optical density at 450 nm was measured. Sera were obtained from healthy lab donors after informed consent was obtained.

Statistical analysis Statistics were analysed with a paired Student’s t-test. Values below P = 0.05 were considered significant.

Acknowledgements This work was supported by The Netherlands Organisation for Scientific Research (NWO) TOP 91206020.

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Supporting information Additional Supporting Information may be found in the online version of this article: Fig. S1. IMPa specificity. Leucocytes were pretreated with 3 mg ml-1 IMPa for 30 min at 37°C and stained with a panel of fluorescently labelled monoclonal antibodies that recognize different cell-surface receptors of leucocytes. Fold inhibition was calculated by dividing the fluorescence of treated cells by that of untreated cells. Data represent the mean value ⫾ SEM of three independent experiments. Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.